Thiostrepton, its salts, and production



May 2, 1961 R. DoNovlcK ETAL THIOSTREPTON, ITS sALTs, AND PRODUCTIONFiled March 1 @.2225 ISEE;

(Ausbau valido) avnveuosev gvwe/wfofra/ RICHARD DONOVICK JOSEPH F.PAGANO JOHN VANDEPUTTE @fw-@M United States Patent()A v Y p 2,982,689 Yrrnos'rREProN, rrs sALTs, AND PRODUCTION Richard Deneviek, Westfield,Joseph F. Pagano, lnamur Brook, and John Vandeputte, Milltown, N J.,assgnors to Olin Mathieson-Chemical Corporation, New York, N.Y., acorporation of Virginia 1,

Filed Mar. 1, 195s, ser. Ne. 491,392 e 7 Claudis. `(C1. 1'6 76s)Y Thisinvention relates to new and useful antibiotics and to methods for theirproduction. More particularly, it :relates to new `antibiotics invarious forms, and to processes for producing them byfermentation, aswell as concentrating, purifying and isolating' them, and producingVtheir salts. In its free form, the new antibiotic is calledthiostrepton. l Y

The antibictica'of this invention isv formed by the cultivation, undercontrolled conditions, of a hitherto undescribed species ofStreptomyces.

'ma MICROORGANISM The microorganism useful for the preparation ofthiostrepton is a strain of Streptornyces isolated from a` sample ofdesert soilobtained lin'New Mexico, United States of America. Acultureof the living organism has been de posited andlmade a part of thestock culture collection of the Rutgers Institute of Microbiology (NewBrunswick,

New Jrersy), from which it is available, and where it has been assignedthe number 3705 in the Waksman collection. It is hereinafter designatedas Streptomyces azureus.

It is to be understood that the invention is not limited to the use ofthe particular organism described herein, but includes inter alia,mutants produced from the described organism by mutating agents, such asX-rays, ultraviolet radiation, actinophages, and nitrogen mustards.

For isolating and characterizing the microorganism, a portion of thesoil sample vis shaken in sterile distilled water and plated on screenagar medium. This medium contains:

Sucrose gms" 10.0 vCitric acid gms 1.2 (NHuaHPQ. gm 0.4 KCl am. 0.08MgCl2-6H2O gm 04.18 MnC12-4H2O mA 0.036 FeCl3-6H2O gm 0.023 ZnC12 gm0.021 CoC126H20 am. [0.004 Agar om 15.0

istilled Water ml f 1000 The cultures are then tested by streak plateprocedure on yeast beef agar for .antibiotic activity -againstbothlbacteria and fungi. lThe culture does not inhibit any of the2,982,689 Patented May 2, 1961 ice tion is white with smoke-gray areasand light Medici blue spots. Reverse growth is pale olive buff with darkglaucous grey spots. No exopigment produced.

Sabouraud agar slant-Glucose, 40 gm.; neopeptone (partially hydrolyzedpeptone), 10 gm.;l agar, 15 gm.; `distilled water to 1000 ml. Growth isgood, colonies have curled edges, spores are white, reverse growth isochraceous tawny to Mars brown. A brown exopigment is produced.

Soy bean infusion agar.-A 2% soybean meal suspension is boiled for 30minutes and filtered hot. The pH is then adjusted to 7 withV sodiumhydroxide and 0.2% glucose, 0.5% NaCl, and 2% agar is added. lGrowth isgood, colonies have entire edges, spores are white to pale Paynes grey,reversel growth is pale olive buff. No exopigment is produced. l

Henricis agar.-Caseinate or NZ-case (hydrolyzed casein), 5 gm.;glycerol, 5 ml.; K2HPO4, 2 gm.; MgSo4-7I-I2O, trace; agar, 15 gm.;distilled water to 1000 ml., pH 7.0. Growth is good, colonies haveentirev edges, spores are white with dark dullgrey sections, rreversegrowth is blackish brown. No exopigment is produced.

Yeast ,beef agar.-Beef extract, 1.5 gm.; yeast extract, 3 gm.;v peptone,6 gm.; dextrose, 1 gm.; agar, 15 gm.; distilled water to 1000 m1. Growthlis slight, colonies have entire edges, spores are white, reverse growthis avellaneous. No exopigrment produced. l

The culture liquiiies gelatin and produces acid curd in milk,.but doesnot produce Vindole or reduce nitrate to nitrate. Y p

The organism is capable of utilizing the following carbon sources inbasal medium containing(NHaQzSO.,l as a source of nitrogen: arabinose,rhamnose, xylose, glucose, galactose, fructose, mannose, lactose,maltose, sucrose, dextrin, inulin, rafnose, starch, glycerol, inositoland mannitol. Growth is supported poorly by salicin, sorbitol, sodiumcitrate and sodium acetate. Growth is not supported by dulcitol,ammonium formate, ammonium oxalate, and ammonium tartrate. In a basalmedium containing starch =as 4a source of carbon the following nitrogensources will support growth: ammonium sulfate, sodium nitrate, sodiumnitrite and asparagine. Growth is supported poorly by l-tyrosne andd,ltryptophane. Growth i is not supported by acetamide.

THE ANTIBIOTIC Streptomyces azureus has been found to produce a mixtureof antibiotics. When grown in a suitable medium, at least threedifferent antibiotics are produced. When the mycelium is separated fromthe broth by filtration, one of the antibiotics -is present primarily inthe mycelium mat whereas the other two antibiotics are presentprincipally in the filtrate. The antibiotic with which this invention isprimarily concerned is the one that is extracted from the mycelium mat,which has been assigned the name thio strepton.

In order to form thiostrepton, Streptomycesazureus is grown at lasuitable temperatureqof from 20 CV. to 35 C., preferably `about 25 C. to27 C., under submerged aerobic conditions inan aqueous nutrient mediumcontaining an assimilable, fermentable carbon and energy source and anassimilable nitrogen source. Suitable sources of carbon, as indicatedabove, include carbohydrates such as: starch, dextrin, and sugars suchas maltose, lactose, and glucose; glycerol; lipids; fats; etc. Suitablenitrogen sources include organic nitrogen source, such as asparagine andsoybean mealLas well asninorganic sources of nitrogen, such asammoniumsulfate or sodiumnnitrate. The fermentation is carried out forabout 72 to 168 hours at a vpH in the range of about 6 to 8. At the endof this period of time, la substantial lamount of thiostrepton has beenformed (as shown by bioassays), as more fully disc-losed in theexamples.

After growth has been completed, thiostrepton is isolated` from theIwhole broth by separating the mycelium cake from the broth by filteringor centrifuging. Thiostrepton is then extracted from the mycelium'cakeby means of :a suitable organic solvent, such as chloroform, dioxane,kan N,Ndi(lower alkyl) lower alkano'ic' acid amide (e.g.N,Ndimethylformamide or N,Ndimethyl acetamide) or benzyl alcohol.yThiostrepton isV a weak base which -forms salts with acids,particularly mineral acids (e.g. hydrochloric acid or sulfuric acid)when reacted therewith in 'an organic solvent such as an alcohol (eg.ethanol). The antibiotic also forms complexes with alkaline earth metalsalts (e.g. calciurnchloride ormagnesium chloride) when reactedtherewith in an organic solvent such as an alcohol (e.g. methanol). Boththe acid-addition salts and alkaline earth metal complexes arehydrolyzed whencontacted with water. l Y

The following examples illustratersnitable methods for preparing,purifying Iand forming derivatives of the antibiotics of this invention.

Exampfle 1.-Shake flask fermentation of Streptomyces azureus Germinationstage- A loopful of a culture of Streptomyces azureus is transferredto a500 m1. ask containing .the following medium:

Soybean meal g 15 Dextrose g 30 OaCO3 Y g 2.5 NaCl g 1.0 Tap Water ml1000 Theiiask is incubated for two days at25 C. on a reciprocatingshaker (140 strokes per minute, 1% inch throw).

Fermentation stage.-A 10 ml. inoculum prepared in the germination stage`is transferred from the germination ask to each of five 1 liter flaskscontaining a medium consisting of:

Soybean meal g Dextrose g y 20 CoCl26H2O g 0.005 CaCO3 g 1.0 Tap waterrnl 1000 The flasks are incubated for seven days at 25 C. on areciprocating shaker. At the end ofY seven days, centrifuged neutralbroth samples are tested for activity against Micrococc'us pyogenes var.aureus and Mycobacterium tuberculosis BCG with the' following resultszPOTENCY IN DILUTION UNITSk M. pyogeues M. tuberculosis var. aureus BCGExample 2.-Tank fermentation of Streptomyces azureus Soybean mealDextrose g. 20 CaCO3 g 5 NaCl g l Tap water ml 1000 which has beensterilized for 3o minutes at 121 c. after is added the contents of thegerminator.

4 adjustment of pH to 7.0 to 7.2 with sodium hydroxide, is placed aloopful of the growth from the inoculum source. The culture is incubatedAfor 72 hours at 25 C., at the end of which time 10 m1. of the cultureis transferred to a second ml. portion of medium in a second flask. Thissecond culture is incubated lfor 48 hours at 25 C., at the end of whichtime the contents of the second flask are transferred to a 1000 ml.portion of the medium in a 4-liter flask. This third culture isincubated for 48 hours at 25 C., and then transferred to a IG-gallongerminator containing' 25 liters of a medium which has been previouslysterilized for- 15 minutes! at 121 C. and consists of:

Soybean meal g 750 Glucose g 1000 CaCO3 Q 25 Tap water litersv 25 (pHadjusted to 7.0 with NaOH before andafter sterilization.)

3.0% soybean meal 3.0% glucose 0.5% CaCO3 0.25 Foamrex S (Socony VacuumOilV Company Inc.

' brand of a stable wax emulsion), or other defoame'r AdjustpH to 7.0with NaOH before sterilization TheA fermentor is aerated and agitatedfor 56' to 108 hours atl 25 C. under. a gauge pressure of 10 p.s.i.

At the end of 50 to 80 hours, the potency reaches a maximum of` 10,000to 16,000 dilution units/ml. (against Micrococcus pyogenes var. aureus)and remains substantially constant thereafter.k 'I'he pH of thefermentation broth remains substantially constant, inthe range of 7.0 to7.4, during the fermentation period.

Thiostrepton may then be separated from theV whole broth by theprocedures illustrated in the following examples.

Example S12-Extractionl of thiostreptonl from mycelium Then'eutralVwhole broth is filtered and the cake dried as muchas possible in apress. The wet lter cake is then placed in a volume ofchlorofrmjust'larg'e enough to yield agood slurry (l2-15 gallons per 100lbs. ofwet cake) and agitated vigorously for one-half hour; The mixture isfiltered, Athe chloroformY separated' froml the water present` andthechloroform retained'. The cake is then extracted with a second volume ofchloroform (.8-10 gallons per 100 lbs. of wet cake), iilter'ed'ftheuchloroform separated and combined with the first extract. The combinedchloroform extracts are then distilled under Vacuum (25." C, maximum pottemperature) to 1/50L the original volume. YTo" this concentrate isadded volumes of hexane. After standing for 1 hour, the precipitate isfiltered olf, washed with acetone and dried'.

The precipitate (A) contains 70-90% pure thiostrep'- ton, and can befurther purified and crystallized by dissolving it in dioxane (l g./ 10ml.)", adding carbon (2% w./v.), warming the mixture with stirring to`50 C., then filtering and adding 5 volumes of Wat'erslowly to theltrate. The crystalline precipitate sorobtained is fairly pure but isstill somewhat colored. j It can b'erfurther purified and a whiterproduct obtained',by'redissolving it in dioxane (l g./ 15 mL), filteringand adding to theiil'- trate slowly 5 volumesofv a 50%' aqueous'methanol solution. The mixture is allov'ved to stand overnight at roomassess@ v temperature and then the crystalline precipitate ofthiostrcpton is separated;l washed with acetone and dried. The productis a white or very light yellow, uniformly crystalline material whichassays 100,000-ll0,000 du./ mg. (Mcrocaccus pyogenes var. aureus). Theyields in each of the crystallization and recrystallization steps arebetween about 75 and 80%.

Two additional active materials are also present in small quantities inthe-whole broth. One of these materials may be obtained from the firstcrude precipitate (A) by slurrying itin acetone. This particularmaterial is soluble in acetone and can be isolated therefrom byprecipitation with water or evaporation of the acetone solvent todryness. The bioassay of this. partially purified matcrial is of theorder of 6,0008,000 du.'/mg. (Micrococcus pyogenes var. aureus).

Another material present can' be recovered by crystalliiation of themother liquors of the second precipitation of thiostrepton. It hasgreater solubility than thiostrepton and. is only recovered from therecrystallization or- Ultraviolet spectrum.- The ultraviolet absorptiony shoulders of crystalline thiostrepton in methanol are loganic motherliquors after addition of much Water. The l in vitro bio-potency of thecrude material is of the order of l5,000-20,000 du./mg. (Micrococcuspyogenes var. aureus).

Example 4.--Preparaton of the hydrochloride salt of thiostneptonThehydrochloride of thiostrepton is formed by slurrying the crystallineor crude thiostrepton in an alcohol such as methanol, and then adding anequivalent of hydrochloric acid as a concentrated aqueous solution. Thethiostreptondissolves; indicating formation of a salt. The hydrochloridecan beprecipitated from this solution by adding ether orfethyl acetate.The hydrochloride is unstable asy the solid or in the original methanolsolution, the activity falling olf rapidly. If water is added to thesolid or to the alcoholsolution, the salt is immediately hydrolyzed andthe free base obtained.

Other acid-addition salts, such as the sulfate, can be prepared in the`same way as the hydrochloride, and have the same general properties.

Example 5 .-Preparation of the calcium chloride complex of thiostreptonThe calciumchloride complex is formed by slurrying thiostrepton base ina 0.5% methanolic calcium chloride solution at a concentration of 4-5mg. per ml. The base slowly dissolves, indicating complex formationsince in the absence of calcium chloride it will not dissolve. Whensolution is complete, the complex is precipitated by addition of etheror ethyl acetate. Addition of water to the solid or to theoriginal-methanol solution will cause the complex to immediatelyhydrolyze, yielding the free base.

CHEMICAL AND PHYSICAL PROPERTIES` OF THIOSTREPTON Crystallinethiostrepton has the following physical and chemical characteristics:Melting point.-Darkens at about 325 with decomposition at about246-256"C.

Solubilty.-Good solubility in dioxane, chloroform, N,Ndimethylformamide, N,N dimethylacetamide and benzyl alcohol. Soluble tothe extent of 100-200 mcg./ ml. in a1kano1s,".such as methanol; ethanol,isopropanol;

C. and melts ask cated at the following wave lengths: .Y

Mme) 240 Infra rea spectrum (see figure of the drawing).-The infraredabsorption peaks and shoulders (sh) are located at the followingfrequencies and wave lengths:

PJM) Kol) 3. 04 8, Q6 5. 77 9. 14 6. 03 9. 43 6. 13 9. 68 (Sh) 6. 33 9.80 6. 52 (Sh) 10. 16 6. 62 10. 30 6. 73 10. 52 7. 04 (Sh) 10. 70 7. 2710. 86 (Sh) 7. 32 (Sh) 11. 20 7. 42 (Sh) 11. 68 7. 64 12. 38 7. 80 12.82 7. 88 13. 00 (Sh) 8. 04 (Sh) 13. 15 8. 31 13. 52 8. 58 (Sh 13.70 8.68 (Sh 8. 79- v BIOLOGICAL PROPERTIES 0F "m10srREPToIsI'A Thiostreptonpossesses a wide antibacterial spectrum against many bacteria (andviruses), as indicated by the partial listing following:

Minimal InvA erobacter aerngeue s 50 Escherichia M71 50l Pseudomonasaeruginosa 50 Salmonella schottmulleri..- 50 Shigella sonnet'- 60Klebsiella pneu 50 Proteus vulgaris 50 Salmonella tuphnsrl 30 f l'dysenteriae 30 Further tests have been conducted ineggsvmice, rats,

'i rabbits, and cats to determine the toxicity andv effectiveness ofthiostrelgston y To determine the in vivo activity of thiostreptonagainst c V8 cated in Table 3. For comparison, vtests were also'runusing the potassium salt of penicillin G instead-ofthe antibioticV ofthis invention. n Y j TABLE 3 Activity vs. M. puoi/eues var.

aureus Antibiotic Mode of` Administration Penicillin-sen-Penicillinsitive y resistant l Strain No.- 5 Strain N o. 376 2 DST 1Sr/T DST l S/T Saline control. Average survival'time=20 hours;Survived/Total (S/T) =0/10 100 mcg. antibiotic+1,000 mcg.N,N-dimethy1acetamidc (DMA) Y 222 10/10' 220 10/10 in 0.5 ml.physiological saline solution. i v 50 mcg. antibiotic-F500 mcg. DMA in0.25 m1. physiological 200 9/10 109 5/10 saline solution. Y S 25 mcg.antibiotic-F250 mcg DMA in 0.125 ml. physiological f 89.` 4/10 42 2/10Thiostrepton saline solution. l n

'"f 100 mcg. antibiotici-1,000 mcg. dioxane in 0,5 m1. physiological 190v 8/10 163 7/10 saline solution. y t Y 50 mcg. antibiotic-l-500 mcg.dioxane in 0.25 mi. physiological 44 2/10 0v v 0/9' saline solution. t25 mcg. antibiotic-|1250 mcg. dioxane in.0.l25 m1. physiological 32.1/10 0 0/10 saline solution. g 60 mcg. antibiotic in 0.5 ml..physiological saline solution 155 L 7/10 0 0/10 Penicillin G potassium30 mcg. antibiotic in 0.25 ml. physiological saline solution--. 123 5/100 0/9 15 mcg. antibiotic in 0.125 m1. physiological saline solution 1336/10 0 0/10 l DST=Diierence in survival time over controls (in hours).

. 2 Resistant to 100 units of penicillin in vitro. meningopneum-onitisvirus in eggs, seven-day old embryonated eggs were infected with 100LB50s of a standardized meningopneumonitis virus inoculum viathe yolksac. The eg-gsrwere then treated one day afterinfection via the' yolksac with thiostrepton dissolved in dimethylacetamide (1:10 ratio ofantibiotic to dimethylacetamide) at the levels indicated in Table 1.

TABLE 1 Average Diier- Survivors/ Survival encein Total(after P13502Level tested (mg/egg) Time Survival days) (mg/egg) (hours) Time (hours)1 Control (dimethylacetamide) S A 219 +61 213 -lf-55V 189 +31 Y TABLE 2'Y Percentage Level Tested (mog-.lmouse-in 0.5 ml. of solution) Survival(percent) Control (Sahne solution) 0 40 Y l Y Y 100 20v i Y. 10o 1n t 2065 The foregoing tests demonstrate the particular eifectiveness ofthiostrepton in vivo against micrococcic and streptococcic infections.

To determine the acute toxicity of thiostrepton in mice, a 10% solutionof the antibiotic in N,Ndimethylacet amide was diluted 1 in 20 withdistilled Water' and administered intravenously (0.1 ml./5 sec.) intothe mice. The LDO wasfound to be about 41.7 mgJkg. and the estimatedLDgwas 23.1 mg./kg. Ithas also been determined that a single intravenousinjection of 5 mg. of thiostrepton/kg. has no untoward effect on testanimals such as cats, rats, and rabbits.

Thiostrepton is an eifective anti-infective medicine having the samegeneral antibiotic spectrum as penicillin and thus can be used againstgram positive coccal infections.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

We claim:

l. A process for producing a thiostrepton-containing antibioticpreparation which 'comprises cultivating a strain of Streptomyces aureusin an aqueous nutrient medium comprising an assimilable, fermentablecarbon and energy source and an assimilable nitrogen source, underaerobic condtions until substantial activity is. imparted to said mediumand recovering the antibiotic from said4 medium.

2. A substance eifective in' inhibiting the growth of gram-positivebacteria, selected from the group consisting of `thiostrepton and thesalts thereof, said thiostrepton being a weakly basic substance havingthe following elementary analysis: C=51'.75%, H=5.3'0%, S=9.22%,N=1-5.84%, O=17.89%; has an antibacterial spectrum including thelfollowing bacteria:` Micrococcus pyogenes Var. aureus, Streptococcuspyogenes C203, Bacillus subtilis, Streptococcus faecals, Lactobacillusacdophilus, Clostridium septcus, Dyplococcus pneumonae type 3,Corynebacterium diphtheriaeand Mycobacterium tuberculosis var. bov'z's(BCG) possesses a crystalline structure in the pure state;is-substantially soluble in dioXane, chloroform, \N,Ndimethylformamide,N,N- dimethylacetamide and benzyl alcohol, and relatively insoluble inwater and the lower alkanols; darkens at about 235 C. and melts at about24o-256v C. with decomposition; has an absorption spectrumrneasured inmethanolic HCl with shoulders at the following4 wave lengths: 240, 280,and 305- mil1imicrons;. andy infrared spectrum 9 when suspended inhydrocarbon oil in solid form substantially as shown in the drawing.

3. Thiostrepton, as described in claim 2. 4. A salt of thiostrepton, asdescribed in claim 2.

5. vA hydrochloride of thiostrepton, as described in 5 claim 2.

6. A sulfuric acid salt of thiostrepton, as described in claim 2.

7. A calcium chloride complex of thiostrepton, as described in claim 2.

References Cited in the tile of this patent UNITED STATES PATENTS OTHERREFERENCES Leach Sept. 30, 1952 15 Pages UNITED `STATES PATENT OFFICECERTIFICATE OF CORRECTION Patent No, 2,982,689 May 2, 1961 i RichardDoxiovick et al.

It is hereby certified that errorappears in the above numbered patentrequiring correction and that the said; lLetters-s Patent should read as'corrected below.

Column 1, line 49, right-hand column of the table, for "04.18" read0.418 column 3, line 21, for "suitable" read suitable column 8, line 49,for -."aureus" in italics, read azureus in italics.

Signed and sealed this 3rd day of October v1961.

(SEAL)v Attest:

ERNEST w. SWIDER DAVID L. LADD Attesting Officer Commissioner oflPatents USCOMM-DC

2. A SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF GRAM-POSITIVEBACTERIA, SELECTED FROM THE GROUP CONSISTING OF THIOSTREPTON AND THESALTS THEREOF, SAID THIOSTREPTON BEING A WEAKLY BASIC SUBSTANCE HAVINGTHE FOLLOWING ELEMENTARY ANALYSIS: C=51.75%, H=5.30%, S=9.22%, N=15.84%,O=17.89%, HAS AN ANTIBACTERIAL SPECTRUM INCLUDING THE FOLLOWING BACTERIAMICROCOCCUS PYOGENES VAR. AUREUS, STREPTOCOCCUS PYOGENES C203, BACILLUSSUBTILIS, STREPTOCOCCUS FAECALIS LACTOBACILLUS ACIDOPHILUS, CLOSTRIDIUMSEPTICUS, DYPLOCOCCUS PNEUMONIAE TYPE 3, CORYNEBACTERIUM DIPHTHERIAE ANDMYCOBACTERIUM TUBERCULOSIS VAR. BOVIS (BCG), POSSESSES A CRYSTALLINESTRUCTURE IN THE PURE STATE, IS SUBSTANTIALLY SOLUBLE IN DIOXANE,CHLOROFORM, N,N-DIMETHYLFORMAMIDE, N,NDIMETHYLACETAMIDE AND BENZYLALCHOHOL, AND RELATIVELY INSOLUBLE IN WATER AND THE LOWER ALKANOLS,DARKENS AT ABOUT 235* C. AND MELTS AT ABOUT 246-256* C. WITHDECOMPOSITION, HAS AN ABSORPTION SPECTRUM MEASURED IN METHANOLIC HCIWITH SHOULDERS AT THE FOLLOWING WAVE LENGTHS: 240, 280, AND 305MILLIMICRONS, AND AN INFRARED SPECTRUM WHEN SUSPENDED IN HYDROCARBON OILIN SOLID FORM SUBSTANTIALLY AS SHOWN IN THE DRAWING.